By analysis of thymidine uptake during the first 4 h of incubation and by examining the responsiveness of unfractionated mouse spleen cells upon mitogenic or allogeneic stimulation, some serum factors of major importance for in vitro cultivation of lymphocytes have been examined. Albumin and l-alanine are essential for the maximal preservation of the in vivo-initiated lymphocyte activity during the first hours of incubation in vitro. Transferrin plays a major role in the lymphocyte proliferation, induced by mitogens in vitro, and, finally, zinc and selenium exert a clear enhancing effect on the response to an allogeneic stimulation. The AATSZ medium (RPMI 1640 enriched with l-alanine, albumin, transferrin, zinc chloride, and sodium selenite) enables a proliferation of the same magnitude as or higher than FCS medium. The kinetics are the same, and the cell viability is comparable, but standardization is much simpler with AATSZ. This is primarily because FCS binds some mitogens and contains inhibitors. Consequently, the standardization of such a culture system is dependent on variations from serum batch to serum batch. On the other hand, the current composition of the AATSZ medium promotes the sticking capacity of T lymphocytes and does not support growth of all lymphoid cell lines. Consequently, this defined medium, although not yet suitable as optimal medium for all lymphocyte functions, can advantageously be used for short-term studies of most murine lymphocyte functional and cooperation studies in vitro.